JP4843792B2 - Antigen drug vehicle enabling transmucosal and transdermal administration, method for inducing mucosal immunity using the same, mucosal vaccine and DDS - Google Patents

Description

  The present invention relates to an antigen-drug vehicle that enables transmucosal administration and transdermal administration, and more specifically, an antigen-specific secretory immunoglobulin characterized by using the vehicle for a desired antigen or drug. The present invention relates to a mucosal immunity induction method, mucosal vaccine, allergy prevention / treatment, and drug delivery system that preferentially and effectively produces A.

Conventional inactivated vaccines and toxoids are known to have the following disadvantages:
(1) Poor protection against natural infection route Whereas the vaccination route is subcutaneous or intramuscular, the natural infection route such as bacteria or virus is the mucous membrane of nasal cavity, trachea, intestinal tract, etc. Different from root. It is desired to achieve infection protection by the inoculation route in accordance with the actual condition of natural infection, particularly prevention of infection in the mucosa by vaccine administration via the mucosa.
(2) Low mucosal immunity In vaccine recipients, immunoglobulin G (hereinafter abbreviated as “IgG” or “IgG antibody”) is mainly produced in the blood to induce humoral immunity. However, immunoglobulin A responsible for mucosal immunity (hereinafter abbreviated as “IgA” or “IgA antibody”) is hardly produced and mucosal immunity cannot be expected. The necessity and effectiveness of IgA antibody are as follows: IgA antibody is a mucous membrane that is the gateway to infection of respiratory tracts such as nasal cavity and trachea by droplets and air, and oral infection of intestinal tract. Is responsible for mucosal immunity and plays an extremely important role in clinical immunity. In addition, IgG antibodies are highly specific for antigens, have a narrow spectrum of protection against infection and are almost ineffective at protecting against antigen-mutated pathogens, whereas IgA antibodies are cross-immune, ie cross-neutralizing. Because of its activity, it has a broad spectrum of protection against infection and protects against infection with mutant antigens.
(3) Necessary cost for additional vaccination Since the IgG antibody produced is low only by the first vaccination of the first immunization and a reliable effect cannot be expected, one or more additional vaccinations based on the status of subsequent IgG antibody possession It is necessary to increase blood IgG antibody titer by inoculation, so-called booster inoculation. Therefore, it is necessary to repeat costs and labor, and is effective for elderly people, adults and school children who are blessed with booster vaccinations. Is occasionally seen.
In other words, conventional inactivated vaccines, toxoids, etc. have the effect of inducing mainly the production of IgG antibodies in blood and increasing humoral immunity in recipients, and their effectiveness has been confirmed. . However, since the ability to induce IgA antibody production or mucosal immunity is low, there are limits to the functions and effects sufficient to protect against natural infection. From this situation, many attempts have been made from various aspects so far in order to eliminate the drawbacks of conventional vaccines. For example, qualitative or quantitative improvement of vaccine antigens, production of live vaccines to replace inactivated vaccines, development of new inoculation routes and mucosal vaccines, screening for adjuvants that promote and sustain humoral immunity, mucosal immune adjuvants Trials specified in the development of the. However, development of a safe and effective mucosal vaccine has not yet been achieved.
The development of mucosal vaccine will be described below.
(1) Increasing the vaccine antigen An attempt has been made to increase the vaccine antigen to be inoculated subcutaneously or intramuscularly and to increase the amount of IgG and IgA antibodies secreted into the mucosa. For example, methods have been attempted in which neuraminidase of the viral membrane protein is added to and mixed with a conventional inactivated influenza vaccine to increase antibody production, or MF59 is added and mixed as an adjuvant. However, there are inconveniences such as pain and increased side reactions.
(2) Nasal administration type vaccine In order to protect against infection with IgA antibody, which is considered to be the most effective, a method of inoculating the liquid split antigen directly into the nose was tried, but it has been pointed out that the production amount of IgA is low . Therefore, in order to increase IgA antibody production ability, attempts have been made to increase mucosal immune response, that is, IgA antibody production ability, by adding E. coli heat-labile toxin or cholera toxin as an adjuvant to the split antigen, Because of the current situation where safety is not guaranteed, clinical trials have been discontinued and have not yet been put to practical use.
(3) Live vaccine using a cold-acclimated strain capable of intranasal inoculation A method for inoculating the intranasal cavity with a cold-adapted influenza virus strain that has an optimum temperature for growth of 25 ° C and hardly proliferates at 39 ° C has been put into practical use. However, the mechanism of attenuation of the cold-acclimated parent strain is not clear, and the risk of reversion to toxicity cannot be denied. In addition, because the active ingredient of the vaccine is a live virus, it has a high invasion ability into cells and excellent immunization initialization. However, since mild influenza symptoms occur sporadically, high risk is likely to become severe if infected with influenza. There are some disadvantages, such as being unusable for humans and the elderly.
(4) Other vaccines Although development of vector vaccines using vaccinia virus as a viral vector, live attenuated vaccines using reverse genetics, DNA vaccines using DNA or cDNA itself as an active ingredient has been experimentally promoted. It has not been put into practical use.
Furthermore, the development of an immune adjuvant will be described below.
(1) Immune adjuvant Immune adjuvant is a general term for substances having regulatory activity such as enhancement and suppression of immune response, and substances related to administration forms for the purpose of sustained release and storage of antigens in the inoculated body, The substance is roughly divided into two substances for enhancing or suppressing the immune response. Among these, as the former, adjuvants for administration forms, for example, vaccines and toxoids using aluminum phosphate, alum and the like have already been put into practical use. However, the practical application of an adjuvant for enhancing and enhancing the immune response has not been known yet. For example, BCG live bacteria derived from bacteria, BCG-CWS, endotoxin, glucan, etc., synthesized MDP, levamisole, poly I-poly C, bestatin, etc., and cytokines such as interferon, TNF, CSF, etc. are known. For reasons such as arthritis, rheumatoid arthritis, hyper- _Y globulinemia, adjuvant diseases such as anemia, and insufficient effects, it is considered that safety and efficacy guarantees are necessary for practical use. In addition, a technique (Patent Document 1) that uses a pulmonary surfactant protein derived from a higher animal as an adjuvant is widely known in order to broaden the enhancement of induction of humoral immunity, but its practical use has not been known yet.
(2) Development of adjuvant for mucosal immunization For example, pertussis toxin B oligomer (Patent Document 2), cholera toxin (Patent Document 3), heat-labile enterotoxin B subunit LTB (Patent Document 4) of E. coli, starch particles (Patent Document 5) ), Cholera toxin B chain protein CTB (patent document 6), B subunit of verotoxin 1 (patent document 7), oligonucleotide (patent document 8), interleukin 12 (non-patent document 1), etc. However, it has not yet been put into practical use.
As described above, the necessity of switching from a conventional vaccine inoculated subcutaneously or intramuscularly to a mucosal vaccine that induces the production of IgA antibody in the mucosa, which is a natural virus infection route, is widely and deeply recognized. In particular, as a next-generation vaccine in the 21st century, development and practical application of so-called mucosal vaccine that induces production of IgA antibody, local immunity or mucosal immunity has been awaited all over the world, but has not been achieved yet. The reason is thought to be that a safe and effective adjuvant for imparting a vaccine with a function of inducing IgA antibody production, local immunity or mucosal immunity has not been specified or established.
JP-T-2002-521460 Japanese Patent Laid-Open No. 3-135923 Japanese National Patent Publication No. 10-500102 JP-T-2001-523729 Special table 2002-50452 gazette JP 2003-116385 A JP 2003-50452 A PCT publication WO 00/20039 pamphlet Infection and Immunity, 71, 4780-4788, 2003 Journal of neonatal Nursing, Vol. 10, pp. 2-11, 2004 Biology of the Neonate, Vol. 74 (suppl 1), pp. 9-14, 1998 American Journal of Respiratory Cell and Molecular Biology, 24, 452-458, 2001

This application makes the following a subject. That is, a function of inducing production of IgA antibody, local immunity or mucosal immunity is imparted to a conventional inactivated vaccine, toxoid or the like. Development of safe and effective technology for that purpose. Conversion from conventional humoral immune vaccines to safe and effective mucosal immune vaccines. In addition, prevention and treatment of allergies, and establishment of a transmucosal / transdermal drug delivery system (hereinafter abbreviated as “DDS”) for administering and transporting drugs via the mucosa and skin.
The present invention provides an antigen-specific secretory immunoglobulin A characterized in that as a means for solving the above problems, an antigen-drug vehicle that enables transmucosal administration and transdermal administration, and the vehicle is used for a desired antigen or drug. The present invention provides a mucosal immunity induction method, a mucosal vaccine, an allergy prevention and treatment agent, and a transmucosal / transdermal DDS.
Application and general use of the antigen drug vehicle provided by the present invention brings about the realization and widespread use of mucosal vaccines against various infectious diseases, allergy prevention and treatment agents, and transmucosal / transdermal DDS. The mucosal vaccine is an immunization means adapted to the actual condition of natural infection, and thus exhibits a significantly superior infection protection effect compared to conventional vaccines. Also, the nasal mucosal IgA induced by the antigen drug vehicle results in inactivation of the allergens therein, enabling desensitization. Furthermore, application of the DDS to a wide variety of drugs enhances and promotes the preventive / therapeutic effects of transmucosal and transdermal administration of drugs. As a result, the present invention greatly improves the medical care, health care and hygiene of the entire human race and becomes a long-awaited gospel for workers in the medical care, health care, and hygiene fields of the world. In addition, biologics including conventional and future vaccines, toxoids, etc., and a wide variety of drugs, providing a means to add functions and performance that enable simple transmucosal administration and transdermal administration compared to injection. .

FIG. 1 shows the effect of suppressing influenza virus infection in the nasal cavity (a) and alveoli (b) in various nasal influenza vaccine administrations, and the nasal cavity (c) and alveoli (d) in subcutaneous influenza vaccine administration. * Indicates a significant level (p <0.01) between the group administered with vaccine alone (no AD vehicle or adjuvant) by t test. Example 1
FIG. 2 shows nasal administration (a), subcutaneous injection (b) anti-influenza antibody-producing IgA and IgG amounts in the nasal lavage fluid by influenza vaccine. The white bar indicates the amount of IgA, and the black bar indicates the amount of IgG. (Example 2)
FIG. 3 shows the effect of adjuvants on anti-influenza specific antibody IgA and IgG production in pulmonary lavage fluid by nasal (a), subcutaneous injection (b) influenza vaccine administration. (Example 3)
FIG. 4 shows the effect of PSF-2 and CTB on blood anti-influenza specific antibody production by intranasal (a), subcutaneous injection (b) influenza vaccine administration. (Example 4)
FIG. 5 shows the effect of PSF-2 and CTB on TGF-β1 secretion levels in the nasal cavity (a) and alveolar (b) mucosa by administration of the nasal influenza vaccine. (Example 6)
FIG. 6 shows the effect of PSF-2 and CTB on the production of anti-influenza specific antibodies in the nasal cavity (a), alveoli (b) and blood (c) by the nasal influenza vaccine. (Example 7).
FIG. 7 shows the effect of SPF-2 and CTB on various cytokines secreted from nasal, lung and splenic lymphocytes by nasal influenza vaccine administration. (Example 8).
FIG. 8 shows the effect of PSF-3 on the production of anti-influenza specific antibodies in nasal cavity (a), alveoli (b) and blood (c) by nasal influenza vaccine. (Example 9).

Hereinafter, embodiments of the present invention will be described in detail.
1. Terminology and description of antigen drug vehicle components
(1) Antigen drug vehicle
The vehicle (Antigen and Drug Vehicle, hereinafter abbreviated as “AD vehicle” or “ADV”) is composed of a lipid designed to enable transmucosal administration and transdermal administration of antigens, drugs and the like. It is a complex with protein. The AD vehicle includes the following (a) to (c).
(A) Lung surfactant protein B or fragments thereof (not only natural fragments obtained by proteolytic enzymes, but also artificial fragments obtained by genetic engineering or peptide synthesis, or one or more of the amino acids constituting such fragments are substituted and / or missing. Including lost mutant fragments, etc.).
(B) Lung surfactant protein C or fragments thereof (not only natural fragments obtained by proteolytic enzymes, but also artificial fragments obtained by genetic engineering or peptide synthesis, or one or more of the amino acids constituting such fragments are substituted and / or missing. Including lost mutant fragments, etc.).
(C) Lipids such as phospholipids and fatty acids. Its shape structure is a membrane-like (sheet-like or rolling lipid membrane) having a spine-like or spike-like polypeptide chain on its surface, and the hydrophobic region ends of a plurality of polypeptide chains are inserted into the lipid membrane. It is shaped like a spike and is different from conventional lipid vesicles (liposomes).
When the desired antigen or drug is coexisted, contacted, captured, adsorbed or bound to the antigen-drug vehicle according to the present invention (when placed), transmucosal administration and transdermal administration of such antigen or drug can be performed. . In other words, the vehicle is a vehicle that allows transmucosal and transdermal administration of antigens, drugs, and the like.
A component of AD vehicle, a protein, polypeptide or peptide and lipid used to prepare / manufacture the eagle, ie, pulmonary surfactant proteins B and C, fragments thereof, and at least one amino acid of the fragment peptide are substituted. Details of lipids such as mutated fragments and / or phospholipids and fatty acids will be described later.
(2) Lung surfactant
Pulmonary surfactant has been put into practical use in the treatment of respiratory distress syndrome (RDS) since the mid-1990s, and various preparations derived from humans, cows, pigs and the like have already been widely marketed and commonly used. (Non-patent document 2). In addition, synthetic peptide preparations containing an active domain related to RDS treatment are also commercially available, and design development and synthesis of SP-B and SP-C analogs are also being promoted (Non-patent Document 3). The composition and composition of the pulmonary surfactant is as follows: about 90% lipids (phosphaticel choline 67.3%, phosphatidylglycerol 19.3%, phosphatidylserine 3.2%, other free fatty acids, etc.) and about 10%. % Protein (surfactant proteins A, B, C and D; hereinafter abbreviated as “SP-A”, “SP-B”, “SP-C” and “SP-D”, respectively). The molecular weight is 28-36 kDa for SP-A, 15 kDa for B, 3.5 kDa for C, and 43 kDa for D. SP-A and D are hydrophilic (water-soluble) and lectin-like (membrane associated) Is . SP-B and C are hydrophobic (lipid-soluble) and lipid-binding, and have an ability to fit into a phospholipid membrane and a surface-active effect. Lung surfactant protein genes derived from humans, cows, pigs, etc. are known, for example, full length bases of human SP-B gene DNA in GenBank / NCBI (https://www.ncbi.nlm.nih.gov./) The accession number of the sequence is J02761, that of human SP-C (and SP-C1) is J03890. Hereinafter, human SP-B and C coding regions (CDRs) obtained from NCBI and the amino acid sequences encoded by them are described.
SEQ ID NO: 1: CDR base sequence of human SP-B gene DNA;
SEQ ID NO: 2: human SP-B full-length amino acid sequence decoded from SEQ ID NO: 1;
SEQ ID NO: 3: CDR base sequence of human SP-C gene DNA;
SEQ ID NO: 4: human SP-C full-length amino acid sequence decoded from SEQ ID NO: 3;
SEQ ID NO: 5: CDR base sequence of SP-C1 occupying on human SP-C gene DNA; and
SEQ ID NO: 6: human SP-C1 full-length amino acid sequence decoded from SEQ ID NO: 5.
(3) Protein or peptide used in the present invention
For the preparation and production of the antigen drug vehicle according to the present invention, SP-B and SP- derived from mammals such as humans, cattle, pigs, whales, dolphins, and from fish such as tuna, shark, ray, yellowtail, etc. A combination with C and a combination of SP-B and SP-C1 can be used. For example, human-derived proteins consisting of the full-length amino acid sequences described in SEQ ID NOs: 2, 4 and 6, respectively, a combination of SP-B and SP-C, and a combination of SP-B and SP-C1, Can be used. Further, for example, the hydrophobic (lipid-soluble) region of SP-B and SP-C based on the hydrophobicity value of Kyte-Doolittle, a fragment containing the region, and at least one amino acid of such a fragment peptide is substituted and / or deleted. Mutated fragments etc. can also be used. For example, a natural peptide consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 20 shown below or a peptide obtained by genetic engineering or chemical synthesis, a longer chain peptide containing these peptides, and at least one of such peptides Mutants, synthetic analogs, and the like in which amino acids are substituted and / or deleted can be used. The amino acid numbers are indicated by ordinal numbers sequentially assigned in the C-terminal direction (from the left to the right of the described sequence), with Met occupying the N-terminus of each sequence being the first amino acid.
SEQ ID NO: 7: amino acid sequence Nos. 214 to 225 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 8: amino acid sequence of Nos. 257 to 266 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 9: amino acid sequence of Nos. 29 to 58 of SEQ ID NOs: 4 and 6 (SP-C fragment);
SEQ ID NO: 10: amino acid sequence Nos. 1 to 20 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 11: amino acid sequence of Nos. 102 to 110 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 12: amino acid sequence of Nos. 119 to 127 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 13: amino acid sequence of positions 136 to 142 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 14: amino acid sequence from positions 171 to 185 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 15: amino acid sequence Nos. 201 to 279 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 16: amino acid sequence of Nos. 253 to 278 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 17: amino acid sequence of Nos. 300 to 307 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 18: amino acid sequence of Nos. 317 to 330 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 19: amino acid sequence of Nos. 344 to 351 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 20: amino acid sequence of Nos. 358 to 381 of SEQ ID NO: 2 (SP-B fragment);
SEQ ID NO: 21: amino acid sequence Nos. 24-58 of SEQ ID NOs: 4 and 6 (SP-C fragment).
According to the present invention, at least one selected from SP-B consisting of the amino acid sequences set forth in SEQ ID NOs: 2, 7, 8, 10 to 20 and fragments thereof, and SEQ ID NOs: 4, 6, 9 and 21 Can be used in combination with at least one selected from SP-C (and SP-C1) and fragments thereof.
(4) Lipid used in the present invention
As the phospholipid, it is desirable to use a phospholipid contained in the lung surfactant, such as phosphatidylcholine (lecithin), dipalmitoylphosphatidylcholine, phosphatidylserine, and the like. Others, dipalmitoyl glycerophosphocholine, diacyl glycerophosphoglycerol, phosphatidylglycerol (cardiolipin), dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, Myelin or the like can be used. As the fatty acid, lauric acid, myristic acid, palmitic acid, stearic acid, palmitooleic acid, oleic acid and the like can be used. Furthermore, lipids derived from aquatic animals such as whales, tuna and dolphins with active lung expansion can be used.
(5) Commercial pulmonary surfactant formulation for RDS treatment
According to this invention, safety and effectiveness as an RDS therapeutic agent are approved by the relevant authorities, and commercially available pulmonary surfactants containing hydrophobic or fat-soluble SP-B and SP-C and phospholipids, for example, The trade name Surfacten Infasurf, Curosurf, Humasurf, Exosurf, Alveofact etc. can be used as the AD vehicle. In addition to SP-B and SP-C, commercially available preparations containing hydrophilic or water-soluble SP-A and SP-D include, for example, 1-butanol and these water-soluble proteins SP-A and SP-D. And these are removed to below the detection limit and then used. In consideration of adjustment of the use concentration in the preparation of AD vehicle, it is desirable to use a dry preparation as compared with a liquid preparation.
2. New knowledge underlying the present invention
This invention is in the intense and severe whirl of the background art described above, and is based on the outstanding insight and analysis ability of the leading inventor who has been through trial and error for more than 10 years, and deep academic experience and novel ideas. Based on the following amazing discoveries.
(1) Whereas conventional adjuvants cause inflammation and enhance antigen-presenting ability, four types of active protein components of lung surfactant, which are inherently secreted surfactants in the lung and gastrointestinal mucosa, SP-A A combination of SP-B and SP-C from which A and D are removed from B, C and D, and a phospholipid complex, or SP-B and SP containing these fat-soluble regions (effective regions) When a viral antigen is allowed to coexist with, contact with, or be captured or adsorbed to a complex of a combination of synthetic peptides of both fragments and the lipid membrane (the aforementioned AD vehicle), antigen-presenting cells of the nasal mucosa can be produced without causing inflammation. Activated, viral antigen is efficiently taken up into the cells, and mucosal antiviral IgA production is effectively and preferentially selected without causing induction of IgG production in the mucosa and blood I was found to be guided.
(2) In addition, a combination of SP-B and SP-C and a phospholipid, or a lipid-soluble region thereof (such as a combination of SP-B and SP-C), which has been conventionally used as a safe inactivated vaccine antigen, A combination of a synthetic peptide of both SP-B and SP-C fragments containing the effective region) and a lipid membrane complex (AD vehicle) is added and mixed, while maintaining the high safety of the split antigen and further splitting. It has been discovered that selective induction of secretory IgA production can be achieved with the antigen alone, in which the activation of antigen-presenting cells, which is inferior to that of a live vaccine, is sufficiently enhanced and supplemented.
3. Background to the above discovery
(1) The lead inventor has conducted extensive research to elucidate the mechanism of influenza onset, treatment, and prevention. In the process, the lung surfactant adsorbs and inactivates the tryptase clara of the HA processing protease of the respiratory tract that restricts the hemagglutinin (HA) of the influenza virus membrane protein to express viral membrane fusion activity and infectivity, resulting in inactivation. It was elucidated to block the virus growth cycle.
(2) As a result of subsequent studies, in addition to the above-mentioned effects, lung surfactant selectively activates mucosal antigen-presenting cells to activate immunity against viral antigens and induces secretory IgA. I found that no induction occurred. Furthermore, it was clarified that SP-B and SP-C are important together with lipid components as an active ingredient for enhancing mucosal immunity in pulmonary surfactant, and the effective region of these protein components was identified and the effectiveness of enhancing mucosal immunity was verified. .
(3) Further, as described above, research is conducted from the viewpoint of airway mucosa bioprotective substances and virus infection protection, and pulmonary surfactant secreted in the body induces selective IgA production as a mucosal immune adjuvant derived from the living body. Prove that you are involved.
(4) The above pulmonary surfactant is a physiologically active substance in the living body, and (a) has the property of adsorbing a specific biological substance (Kido H., et al. FEBS Lett. Pulmonary survivant is a potentiogenous inhibitor of proteolytic activation of Sendai virus and influenza A, 322 (29), 115-119, 1992), (b) secreted from alveolar type II cells and Clara cells, and then selectively taken up and metabolized by macrophages (Akira Suwabe, J. Jpn. Med. Soc. Biol. Interface; Surfactant metabolic disorder in alveolar proteinosis, 33, 10-13, 2002), c) an analogue cells were overlaid attention studied to be metabolized, for example, incorporated into antigen presenting cells (dendritic cells).
As a result, among the protein components of lung surfactant, only SP-B, SP-C, and lipid components function as an “AD vehicle” for a mucosal vaccine that selectively induces IgA production. The active ingredient region or mucosal immunity-inducing active domain was revealed to be a peptide consisting of the following amino acid sequences:
SP-B 214-225: Leu Ile Lys Arg Ile Gln Ala Met Ile Pro Lys Gly (SEQ ID NO: 7); and
SP-B 257-266: Leu Leu Asp Thr Leu Leu Gly Arg Met Leu (SEQ ID NO: 8).
In addition, it was clarified that the active ingredient region of SP-C or the mucosal immunity inducing active domain is a peptide comprising the following amino acid sequence:
SP-C 29-58: Cys Pro Val His Leu Lys Arg Leu Leu Ile Val Val Val Val Val Val Leu Ile Val Val Val Ile Val Gly Ala Leu 9
(5) Furthermore, as a mechanism for selective IgA induction, the active ingredient of lung surfactant induces an increase in the expression of MHC Class II, CD40, B7-2 in antigen-presenting dendritic cells and antigens to T-lymphocytes. In addition to effectively performing the presentation, it was clarified that the cytokine TGF-β1 localized in the mucosa was induced to promote class switching to IgA-producing B-lymphocytes.
4). The object of the present invention completed based on the above discovery and its background is as follows.
(1) The first purpose is to establish a mucosal immunization method. By providing "AD vehicle" and its utilization, selective induction of antigen-specific IgA production, which is an effective substance of mucosal immunity, is realized, and induction of safe and effective (no side effects) mucosal immunity and its method are established. To do.
(2) The second purpose is to improve the quality of AD vehicle related to safety, effectiveness and homogeneity by using synthetic peptides. SP-B mucosal immunity-inducing active domain (consisting of the amino acid sequences of SP-B 214-225 and SP-B 257-266) and SP-C mucosal immunity-activating domain (SP-B) C 29-58 (consisting of the amino acid sequence), synthetic analogs thereof, long-chain synthetic peptides containing these amino acid sequences as a part, and the surfactant surfactant lipid component. Provide composite (AD vehicle) to improve the quality of AD vehicle.
(3) The third purpose is to switch from subcutaneous injection of conventional vaccines to transmucosal administration. AD vehicle is used as an inactivated vaccine for respiratory tract virus, such as influenza, SARS, measles, rubella, mumps, etc., and an inactivated vaccine for enteric virus, such as rota, polio, etc. Convert these subcutaneous vaccines into mucosal vaccines.
(4) The fourth object is to provide an AD vehicle that can be used in inactivated vaccines against viral infections other than the respiratory tract and intestinal tract, such as AIDS, hepatitis B, and hepatitis C. That is.
(5) A fifth object is to provide a method that can use an AD vehicle for DNA vaccines, live vaccines, prevention and treatment of allergies, and the like.
(6) The sixth object is to provide an AD vehicle that can be used in transcutaneous inoculation (application, pasting, etc.) as an immunization route capable of inducing IgA other than mucous membranes.
(7) The seventh purpose is to open the road to the use and application of AD vehicles not only for DDS and pharmaceuticals, but also for agriculture, fishery and so on.
The AD vehicle proposed by the present invention differs in performance and action from the adjuvants conventionally used in immunology as follows. That is, conventional adjuvants are usually inoculated subcutaneously or intramuscularly, cause a local inflammatory reaction, attract antigen-presenting cells, B- and T-lymphocytes, and contain foreign substances that exhibit their ability as active ingredients. Furthermore, in order to maintain an inflammatory reaction over a long period of time, mineral oil and metal salts that cause slow release and storage of antigens are used in combination. Moreover, what is known as a conventional mucosal vaccine / adjuvant is a foreign substance such as Escherichia coli heat-labile toxin or cholera toxin as described above, and therefore has a risk of causing harmful effects and side effects. In contrast, the AD vehicle according to the present invention does not cause a local inflammatory response. In addition to being derived from biological components, the active ingredient in the lung surfactant or its active domain is limited, and an effective mucosal vaccine is realized by using such a domain and a low molecular peptide containing the domain region. ing. Therefore, it is extremely safe and non-invasive.
5). According to this invention, the following (1) to (5) are provided respectively.
(1) at least one fragment selected from lung surfactant protein B or a plurality of fragments derived from protein B, at least one fragment selected from lung surfactant protein C or a plurality of fragments derived from protein C, and at least one An antigen-drug vehicle that is a complex of lipids of a species.
More specifically, the AD vehicle comprises the following group I (pulmonary surfactant protein B and natural and synthetic polypeptide groups derived from or derived from the protein B) group II (pulmonary surfactant protein C and natural derived from or derived from the protein C). And a composite polypeptide group) and a group III (a lipid group such as phospholipid and fatty acid), and a total of at least three substances selected from each group.
[Group I] Pulmonary surfactant protein B and a polypeptide comprising the following amino acid sequence described in SEQ ID NO: 2 (the amino acid number is the N-terminal Met as the first amino acid, and sequentially attached in the C-terminal direction from this) No. 1-381 (sequence number 2), 214-225 (sequence number 7), 257-266 (sequence number 8), 1-20 (sequence number 10), 102- 110 (sequence number 11), 119-127 (sequence number 12), 136-142 (sequence number 13), 171-185 (sequence number 14), 201-279 (sequence number 15) ), 253-278 (SEQ ID NO: 16), 300-307 (SEQ ID NO: 17), 317-330 (SEQ ID NO: 18), 344-351 (SEQ ID NO: 19), 358-381 Number No. 20), a polypeptide having at least one of the above amino acid sequences as an active domain, a polypeptide in which at least one amino acid of each of the above amino acid sequences is substituted and / or deleted, synthetic analogs thereof, These sugars or sugar chain-modified products, etc.
[Group II] Pulmonary surfactant protein C and a polypeptide comprising the following amino acid sequence described in SEQ ID NO: 4 (the amino acid number is assigned to the N-terminal Met as the first amino acid and sequentially attached in the C-terminal direction from this) 1): No. 1-197 (SEQ ID NO: 4), 29-58 (SEQ ID NO: 9), 24-58 (SEQ ID NO: 21), consisting of amino acid sequence No. 1-191 of SEQ ID NO: 6 Polypeptide, polypeptide having at least one of the above amino acid sequences as an active domain, polypeptide in which at least one amino acid in each of the above amino acid sequences is substituted and / or deleted, synthetic analog thereof, Etc. modified by sugar or sugar chain, etc.
[Group III] Phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylserine, dipalmitoylglycerophosphocholine, diacylglycerophosphoglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid and other phospholipids, lauric acid, myristic acid, palmitic acid Lipids such as fatty acids such as stearic acid and oleic acid.
Further, in this antigen drug vehicle, the group III is a sheet-shaped or roll-shaped lipid membrane, and each of the plurality of chains of the group I and the group II is implanted in a spike shape with the end of the hydrophobic region inserted into the lipid membrane. Chaining is one preferred aspect of the configuration and shape.
(2) A mucosal vaccine obtained by coexisting, contacting, capturing, or adsorbing an antigen to the antigen drug vehicle of (1) above, and inducing mucosal immunity.
(3) A prophylactic and therapeutic agent for allergy obtained by coexisting, contacting, capturing or adsorbing an allergen to the antigen drug vehicle of (1) above, and inducing mucosal immunity. The effect is, for example, inactivation or desensitization by nasal cavity or nasopharyngeal mucosa IgA of allergens such as Japanese cedar pollen and mites that are sucked in and out.
(4) A transmucosal and / or transdermal DDS obtained by coexisting, contacting, capturing or adsorbing a drug having a medicinal effect on the antigen drug vehicle of (1).
(5) A method for inducing mucosal immunity, comprising administering to the nose or upper respiratory tract a mucosal vaccine obtained by coexisting, contacting, capturing or adsorbing an antigen to the antigen drug vehicle of (1) above.
In the above inventions (2), (3) and (5), mucosal immunity is induced by promoting production of IgA antibody in the mucosal region and further promoting production of TGF-β1 and Th2 type cytokines in the mucosal region. It is made into the preferable aspect to be characterized by these.
6). Hereinafter, embodiments of the present invention will be described.
(1) Composition of AD vehicle
Group I (pulmonary surfactant protein B and natural and synthetic polypeptide groups derived or derived from protein B), Group II (pulmonary surfactant protein C and natural and synthetic polypeptide groups derived from or derived from protein C), and The dry weight% of Group 3 of Group III (lipid group such as phospholipid and fatty acid) is as follows: Group I is about 0.1 to about 6.0% by weight, Group II is about 0.1 to about 0.1% About 6.0% by weight, and Group III is about 88 to about 99.8% by weight. In the preparation of the antigen drug vehicle, the weight% is adjusted so that Group I% + Group II + Group III% = 100%.
(2) Preparation of AD vehicle
The preparation procedure is illustrated below. For example, 2 mg of group I, 2 mg of group II, and 96 mg of group III are weighed (% by weight of group I + group II + group III% = 100%), and these are mixed with 5 ml of isotonic solution, for example, physiological Suspend uniformly in saline or phosphate buffered saline (PBS). The obtained suspension is used as an antigen drug vehicle (100 mg / 5 ml) solution. The vehicle is prepared each time it is used. In addition, an ultrasonic wave, a homogenizer, a mixer, a shaker, etc. can be used for suspension. Since ultrasonic waves tend to cause liquid denaturation (increase in viscosity) due to excessive treatment, it is desirable to use a mixer such as a box mixer (for example, the trade name Vortex mixer).
In addition, about the breakdown of the lipid of group III, for example, a mixture of 71 mg of phosphatidylcholine, 21 mg of phosphatidylglycerol, and 4 mg of phosphatidylserine can be employed (total amount of lipid is 96 mg). In addition, when using a commercially available pulmonary surfactant preparation for RDS treatment in which SP-A and D are removed and SP-B and C are surely contained, the suspension prepared according to the instructions for use is used as it is. Can be used as a vehicle solution.
(3) Preparation of mucosal vaccine
An antigen drug vehicle solution is added to the vaccine stock solution and mixed so that the dry weight ratio A / V of the antigen drug vehicle amount (V) to the antigen amount (A) in the vaccine is about 0.2 to about 5. For example, when a weight ratio of A / V = 1 is adopted for 1,000 ml of a vaccine stock solution having an antigen content of 1 μg / ml, the added amount of the antigen drug vehicle (100 mg / 5 ml) prepared in the above (2) is 50 μl. In addition, in order to mix uniformly, a homogenizer, a mixer, a shaker, a stirrer, etc. can be used.
(4) Preparation of transmucosal / transdermal DDS
It can be prepared by using a drug instead of an antigen in the same manner as in (3) above.

Hereinafter, examples will be shown, and the configuration and effects of the present invention will be specifically described. However, the present invention is not limited only to these specific examples, explanations and descriptions.
The materials, test procedures, etc. used in this example are as follows.
(1) Lung surfactant
Lung surfactant used as “antigenic drug vehicle (AD vehicle)” was prepared by the method of Howgood S, et al.,: Effects of a surfactant-and-cir- sition of citrus rations on the basis of cattle lungs. of surf surfactant lipids.Biochemistry 24,184-190, 1985), or in many cases, this preparation is extracted with 1-butanol and water-soluble protein components SP-A, SP- D sample removed or reduced to below detection limit (Haagsman HP, et al.,: The major lunge surfactant p otein, SP28-36, isa calcium-dependent, carbhydrating binding protein, J. Biol. Chem. 262, 13977-13880, 1987) (PSF-2), and phospholipids mainly comprising phosphatidylcholine and dipalmitoylphosphatidylcholine as a whole. Synthetic peptides containing SP-B and SP-C, which are fat-soluble proteins, containing 40% by weight or more, further containing 10-20% phosphatidylglycerol and 2-5% phosphatidylserine, similar to lung surfactant lipids A standard containing 0 to 3.5% of either or both peptides is used. Among these preparations, a preparation (PSF-3) having both peptides of SP-B and SP-C effective region, a preparation (PSF-4) having a synthetic peptide of SP-B effective region, A specimen (PSF-5) having a synthetic peptide of the effective region of SP-C was also examined. Alternatively, known preparations corresponding to PSF-1 and PSF-2, such as trade name Surfacten, Insurfurf, Curosurf, Humansurf, Exosurf, Alveofact, etc. Is also used.
(2) Animals
Six-week-old female BALB / c mice and 10-week-old Hartley guinea pigs were purchased from Nippon SLC Co., Ltd. (Shizuoka, Japan) and used. All animal experiments were performed at the Infectious Animal House (P2 level) of the University of Tokushima Medical School Experimental Animal Center, and were conducted according to the guidelines of the Animal Experiment Committee of the University of Tokushima Medical School.
(3) Production of split influenza vaccine
Suspension derived from embryonated chicken eggs inoculated with influenza virus A Aichi / 68/2 / H3N2 strain (1 × 10 ⁸ Using a Plaque forming unit (PFU) (provided by Professor Masanobu Ouchi, Department of Microbiology, Kawasaki Medical University), a split influenza vaccine was prepared by the following operation. Β-propiolactone (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to a virus suspension that was dialyzed overnight in 0.004M PBS (Takara Bio Inc., Japan, Tokyo, Shiga). It was added to 8 nM and incubated in an ice bath for 18 hours. Thereafter, β-propiolactone was hydrolyzed by incubating at 37 ° C. for 1.5 hours. Thereafter, Tween 20 (Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 0.1%, and diethyl ether (Wako Pure Chemical Industries, Ltd.) in an amount equivalent to Tween was further added, and the mixture was mixed by inverting at 4 ° C. for 2 hours. The aqueous layer was recovered by centrifuging this solution at 2000 rpm for 5 minutes. Further, diethyl ether was removed from the aqueous layer using an Automatic Environmental SpeedVac System (SAVANT INSTRUMENTS, INC. New York, USA). This was filtered through a Millex 0.45 μm filter (MILLIPORE USA, Massachusetts) and used as an inactivated split influenza vaccine.
(4) Immunization
The split influenza vaccine prepared by the above production method has an effective region of the above-mentioned lung surfactant (PSF-1), 1-butanol extracted lung surfactant (PSF-2), SP-B and SP-C as “AD vehicle” A synthetic peptide and pulmonary surfactant lipid (PSF-3, 4, 5), a known pulmonary surfactant product, or cholera toxin B subunit (CTB, SIGMA USA, MO) was used as a mixture as an immune adjuvant. Lung surfactant or the above-mentioned equivalent preparation was suspended in PBS at the concentration required for vaccine administration and sonicated at room temperature for 5 minutes to obtain a uniform suspension. To this, 0.1 μg of the split influenza vaccine was added to 0.1 μg of the dry weight of the lung surfactant or the above-mentioned equivalent preparation, mixed with a vortex mixer, and allowed to stand at room temperature for 1 hour. Similarly, CTB was adjusted at the time of use and mixed to 0.1 μg with respect to 0.1 μg of the split influenza vaccine (Watanabe I, et al.,: Characteristic of protected responses injured cinnacin cinnacin toxin as a safe Adjuvant (CT112K). Vaccine 2002; 20: 3443-55).
In nasal administration of the vaccine, the above preparation is diluted with PBS to give a 0.1 μg / 1 μl Phosphate buffered saline (PBS) solution equivalent to the dry weight, and this is 1 μl per side per mouse on both sides. Once administered, a total of 2 μl was administered nasally into the bilateral nasal cavity of anesthetized ether. In the conventional subcutaneous injection performed for comparison, the split influenza vaccine was diluted with PBS so as to be a 0.1 μg / 50 μl solution, and this was subcutaneously administered to the mouse neck. The control group received PBS in the same amount as the vaccine solution. After 4 weeks, secondary immunization was carried out in the same manner as the primary immunization, and nasal cavity / alveolar lavage fluid and serum were prepared 2 weeks after the secondary immunization, and virus-specific IgA and IgG measurements and virus were prepared. Used for infection experiments.
(5) Preparation of mouse nasal cavity / alveolar lavage fluid and serum
The vaccinated mice were laparotomized under pentobarbital anesthesia, the trachea was opened, and 3Fr (Atom Medical Co., Ltd., Tokyo, Japan) with an Atom Vein Catheter was inserted into the lung. It was collected. This was repeated 3 times, and a total of 3 ml was used as the alveolar lavage fluid. After collecting the pulmonary lavage fluid, an atom venous catheter was inserted from the incised trachea toward the nasal cavity, 1 ml of physiological saline was injected, and the fluid that came out of the nose was collected. This solution was used as a nasal wash. Furthermore, blood was collected from the heart, and serum was prepared by centrifugation at 5,000 rpm for 10 minutes.
(6) Protein determination
The protein content of nasal lavage fluid, lung lavage fluid, and serum was measured using the BCA Protein Assay Reagent Kit (PIERCE USA, Illinois) (Smith PK. Et al., Measurement of protein biotinch. 150, 76-85, 1985). Absorbance at 562 nm was measured using SPECTRAmax PLUS 384 (Molecular Devices Corporation, California, USA).
(7) Virus infection experiment and evaluation of infectious titer
The same influenza virus strain A Aichi / 68/2 / H3N2 strain as that used for the preparation of the split influenza vaccine was used for infection. After 2 weeks from the end of the second immunization, the mice were anesthetized with ether, and then the suspension derived from the influenza virus-developing hen's egg was placed in both nasal cavities for a total of 7 × 10 ⁴ PFU / 3 μl was added dropwise to infect. Three days after infection, the nasal cavity and alveolar lavage fluid were prepared as described above and used for evaluation of the virus infectivity. Evaluation of virus infectivity was performed using A549 cells (provided by Professor Masanobu Ouchi, Department of Microbiology, Kawasaki Medical University). A549 cells were cultured under conditions of 5% fetal bovine serum / DMEM (Gibco USA, New York). A549 cells were subcultured to 6-well culture plates (Grainer Stuttgart, Germany) to be 100% confluent and replaced with serum-free medium after 24 hours. 500 μl of nasal / alveolar lavage fluid from influenza-infected mice was dropped into each well, and CO ₂ Culturing was performed at 37 ° C. for 12 to 16 hours in an incubator. To this was added 1% PBS of erythrocytes collected from guinea pigs and allowed to stand at room temperature for 5 minutes. This is 1 mM Ca ²⁺ / Mg ²⁺ Cells washed with PBS and aggregated erythrocytes were counted as virus-infected cells, and the virus infectivity was evaluated (Tashiro M., Hamma M .: Pneurotropism of Sendai virus to protease-mediated activation in). lungs.Infect.Immun.39,879-888,1983).
(8) Purification of anti-influenza specific IgA and IgG
Purification of specific anti-influenza IgA and IgG was performed as follows for use as a quantitation standard in ELISA assay. The IgG fraction was purified from lung vaccine of influenza vaccine-administered and virus-infected mice by affinity chromatography using recombinant E. coli-expressed Protein G Sepharose 4B column (ZYMED LABORTORIES INC, San Francisco, USA). Anti-mouse IgA goat IgG (SIGMA) was bound to a BrCN-activated Sepharose 4B column (Amersham Bioscience USA, New Jersey), and the IgA fraction was purified by affinity chromatography using this from the ProteinG flow-through fraction. In order to purify virus-specific antibodies from these IgG and IgA fractions, the inactivated split influenza vaccine used for immunization was bound to a BrCN-activated Sepharose column, and the antigen affinity chromatography using this from the IgA and IgG fractions. Anti-influenza specific IgA and IgG were purified by graphy, respectively. Coupling the split influenza protein as a ligand to the column is 0.1M NaHCO 3. ₃ The binding reaction was performed using a 0.5 M NaCl buffer (pH 8.5), free ligand was removed using 0.1 M acetic acid / 0.5 M NaCl buffer (pH 8.5), and then PBS (pH 7. Neutralization was carried out according to 5). In each affinity chromatography, the affinity binding reaction and free antibody removal were performed with PBS (pH 7.5), and then the specific antibody was eluted with glycine-HCl buffer (pH 2.8). The eluted fraction was immediately neutralized with 0.5 M Tris-HCl buffer (pH 9.0), dialyzed with MilliQ water, lyophilized, and dissolved in PBS before use.
(9) Quantification of anti-influenza antibodies
Anti-influenza IgA and IgG content in nasal cavity, alveolar lavage fluid and serum were quantified by ELISA assay. The ELISA assay was performed according to the method of Mouse ELISA quantification kit of BETHYL LABORATORIES (Texas, USA). 96-well Nunc immunoplate (Nalgen Nunc International USA / New York) Each well was added with 1 μg of vaccine and bovine serum albumin (BSA, SIGMA USA / MO) 100 μl of PBS solution, and solidified at 4 ° C. overnight. It was. Thereafter, the vaccine solution was rinsed three times with a washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0). 200 μl of 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl and 1% BSA was added to each well, and a blocking reaction was performed at room temperature for 1 hour. After rinsing each well three times with a washing solution, a nasal washing solution and a lung washing solution diluted with a sample binding buffer (50 mM Tris, 0.15 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) to an appropriate amount. Alternatively, 100 μl of serum was added and reacted at room temperature for 2 hours. GOT anti-mouse IgA or IgG-horse rAdish peroxidase (HRP) (BETHYL LABORATORIES INC.) Was used as a secondary antibody, and TMB Microwell Peroxidase SubLike System L Went. 100 μl per well, 2M H ₂ SO ₄ The reaction was stopped by adding (Wako Pure Chemical Industries, Ltd.), and the absorbance at 450 nm was measured with SPECTRAmax PLUS 384. As the standard for quantification, the absorbance obtained in the same manner for anti-influenza IgA and IgG 10 ng purified from the lung lavage fluid was used.
(10) Dendritic cell preparation and flow cytometry
Dendritic cells were prepared from the nose, lungs, and spleen collected from each group of mice (4 mice per group) on the second day after the first immunization by the method of Gonzalez-Juarrero M (Charzalez-Juarrero M, Orchar IM .: Characterization. of murine luden dendritic cells infused with Mycobacterium tuberculosis. Infect Immuno 2001; 69: 1127-33). The preparation of dendritic cells from the nose and their collagenase treatment were carried out by a method such as Asanuma H (Asanuma H, et al.,: Characterization of mousse lysated by 19 ol. ). Dendritic cells prepared from each tissue were washed with 1 mM EDTA / PBS, 10 ⁶ FITC conjugated Anti-IA / IE (MHC class II) and PE conjugated Anti-CD40 per cell or FITC conjugated Anti-CD80 (B7-1) and PE conjugated Anti CD86 (B7-2) G1 USA (B7-2) / Ml was added and reacted as a 50 μl 1 mM EDTA / PBS suspension on ice for 30 minutes. The free antibody was removed by washing twice with 1 ml of 1 mM EDTA / PBS, and resuspended in 1 ml of 1 mM EDTA / PBS. Using this, the cell surface modifier was detected by BD FACS Callibur (BD Bioscience).
(11) Quantification of TGF-β1
The secretion amount of TGF-β1 in the nasal cavity and alveolar lavage fluid was quantified by ELISA assay.
ELISA assay was performed using TGF-β1 ELISA kit (BIOSOURCE INTERNATIONAL, California, USA) according to the usage attached to the kit.
(12) Quantification of various cytokines
Quantities of cytokines (interleukin 4: IL-4, IL-5, IL-6, IL-13) secreted from nasal cavity, alveolar lavage fluid, and spleen lymphocytes were quantified by respective commercially available ELISA kits. did.
(13) Preparation of PSF-3 consisting of SP-B and SP-C synthetic peptides and phospholipids
Each peptide of SP-B 253-278 (SEQ ID NO: 16) and SP-C 24-58 (SEQ ID NO: 21) was chemically synthesized by a known method. These peptides were added to a phospholipid membrane (dipalmitoylphosphatidylcholine (75), phosphatidylglycerol (25), palmitic acid (10)) to form a plate-like phospholipid membrane, and AD vehicle PSF-3 was prepared.
Example 1
Comparison of virus growth inhibitory effect of nasal influenza vaccine and subcutaneous injection influenza vaccine
As a nasal vaccine, 0.1 μg of a split influenza vaccine alone, or as an “AD vehicle”, 1-butanol extraction (SP-A, SP-D removal) surfactant (hereinafter PSF-2) 0.1 μg, or CTB 0.1 μg At the same time, 1 μl of each solution was administered to both noses of BALB / c mice as a PBS solution. As a subcutaneous injection vaccine, the same amount of vaccine as the nasal vaccine alone, PSF-2 as an AD vehicle, or CTB as an adjuvant was added, and the whole was subcutaneously administered to the neck of the BALB / c mouse as a 50 μl PBS solution. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. 6.6 × 10 after 2 weeks of secondary immunization ⁴ PFU influenza virus was infected nasally as a 3 μl PBS solution. Three days after infection, the mice were sacrificed, and nasal and alveolar lavage fluids were prepared and used to evaluate the viral infectivity titer (n = 15 to 20; mean ± SE; *, significance level by t-test was vaccinated. P <0.01 for the group.
As shown in FIGS. 1 (a) and (b), in the case of nasal influenza vaccine administration, the growth of influenza virus in the nasal cavity and lung lavage fluid is also suppressed by the vaccine alone administration, but PSF-2 and CTB are The effect was significantly enhanced to suppress the growth of influenza to almost completeness and ensure the effectiveness of the vaccine. Although not shown in the figure, even if PSF-1 or PSF-3 is used instead of PSF-2, a slight effect is obtained. The same phenomenon was confirmed for PSF-4 and -5, but the effect was reduced.
Although not shown in the figure, even if PSF-2 and CTB were administered nasally at the time of primary immunization and secondary immunization alone, no effect of suppressing virus growth was observed. The effect was determined to be a vaccine effect enhancement effect.
On the other hand, as shown in FIGS. 1 (c) and (d), the influenza virus titer (PFU) in the nasal cavity and lung lavage fluid decreased significantly even with the vaccine alone, and the effect of the vaccine was confirmed. However, the effect of enhancing the vaccine action of PSF-2 and CTB was not observed. In other words, in the case of subcutaneous administration, it was estimated that PSF-2 and CTB had little or no effect on immune enhancement. In this experiment, although not shown in the figure, even when PSF-2 and CTB were each administered subcutaneously at the time of the first immunization and the second immunization, no effect of suppressing the virus growth was observed. Although not shown in the figure, even if PSF-1, PSF-3, PSF-4, or PSF-5 was used instead of PSF-2, the effect of enhancing the vaccine action was not recognized.
Example 2
Effects of PSF-2 and CTB on production of anti-influenza specific antibodies (IgA, IgG) in nasal lavage fluid after influenza vaccine administration by nasal (a) and subcutaneous injection (b)
In the method described in FIG. 1, 0.1 μg of split influenza vaccine alone as a nasal vaccine, or 0.1 μg of PSF-2 as an AD vehicle, or 0.1 μg of CTB as an adjuvant, and both noses of a BALB / c mouse as a PBS solution A total of 2 μl of 1 μl was administered. As a subcutaneous vaccine, the same amount of vaccine as the nasal vaccine, PSF-2, CTB was administered subcutaneously to the neck of the BALB / c mouse as a 50 μl PBS solution. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. 6.6 × 10 after 2 weeks of secondary immunization ⁴ PFU influenza virus was infected nasally as a 3 μl PBS solution. Three days after infection, the mice were sacrificed, nasal washes were prepared, and these were used to evaluate the viral infectivity titer (n = 15 to 20; mean ± SE; *, t test significant level relative to the vaccine administration group P <0.01).
The results are shown in FIGS. 2 (a) and 2 (b). Anti-influenza-specific IgA is selectively produced in the nasal cavity and increased in the lavage fluid by the nasal split influenza vaccine, but this specific IgA amount is significantly increased in both PSF-2 and CTB. I let you. On the other hand, in the case of subcutaneous injection, an increase in specific IgA in the nasal lavage fluid by the split influenza vaccine alone was observed, but the level was lower than that of nasal administration. In the case of subcutaneous injection of vaccine, neither PSF-2 nor CTB immune enhancing effects were observed in IgA and IgG production. Although not shown in the figure, even if PSF-1 or PSF-3 is used instead of PSF-2, the same effect as PSF-2 is obtained. The same phenomenon was confirmed for PSF-4 and -5, but the effect was attenuated.
Example 3
Nasal administration (a), subcutaneous injection (b) Effects of PSF-2 and CTB on anti-influenza specific antibody (IgA, IgG) production in lung lavage fluid by influenza vaccine
Inject 0.1 μg of split influenza vaccine as a nasal vaccine alone, 0.1 μg of PSF-2 as an AD vehicle, or 0.1 μg of CTB as an adjuvant, and administer 1 μl to both noses as a PBS solution in a total volume of 2 μl. It was. As a subcutaneous vaccine, the same amount of vaccine as the nasal vaccine, PSF-2, CTB was administered subcutaneously to the neck of the BALB / c mouse as a 50 μl PBS solution. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. 6.6 × 10 after 2 weeks of secondary immunization ⁴ PFU influenza virus was infected nasally as a 3 μl PBS solution. Three days after infection, the mice were sacrificed, alveolar lavage fluids were prepared, and the effects of the antigen drug vehicle on the production of anti-influenza specific antibodies IgA and IgG were examined. (N = 15-20; mean ± SE; *, significance level by t test is p <0.01 with respect to the vaccine administration group).
As shown in FIGS. 3 (a) and 3 (b), the effect of promoting the production of PSF-2 and CTB against anti-influenza-specific antibodies produced in the lung lavage fluid is remarkable as in the case of nasal administration of the vaccine. There was no big difference between them. This immunopotentiating effect was specific to IgA, and no effect was observed on IgG. In the case of subcutaneous injection, IgA increased during lung lavage, but the immune enhancing effect of PSF-2 and CTB was not observed as in nasal lavage fluid. Although not shown in the figure, increases in virus-specific IgA and IgG during pulmonary lavage were observed even when PSF-2 and CTB were administered nasally or subcutaneously during the first and second immunizations, respectively. However, the effect of PSF-2 and CTB was determined to be an effect of enhancing vaccine action. Although not shown in the figure, even if PSF-1 or PSF-3 is used instead of PSF-2, a slight effect is obtained. The same phenomenon was confirmed for PSF-4 and -5, but the effect was attenuated.
Example 4
Effects of PSF-2 and CTB on intranasal administration (a), subcutaneous injection (b) production of anti-influenza specific antibodies (IgA, IgG) in blood by influenza vaccine
Inject 0.1 μg of split influenza vaccine as a nasal vaccine alone, 0.1 μg of PSF-2 as an AD vehicle, or 0.1 μg of CTB as an adjuvant, and administer 1 μl to both noses as a PBS solution in a total volume of 2 μl. It was. As a subcutaneous injection vaccine, the same amount of vaccine as nasal vaccine, PSF-2, CTB was administered subcutaneously to the neck of the BALB / c mouse as a 50 μl PBS solution. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. Two weeks after the second immunization, the mice were sacrificed, blood was collected from the blood, serum was prepared therefrom, and the amount of anti-influenza antibody expression was quantified using these (n = 15-20, mean ± SE).
As shown in FIGS. 4 (a) and 4 (b), the production amounts of anti-influenza antibodies IgA (white bars) and IgG (black bars) in blood are slightly increased in IgA and IgG in the case of nasal vaccine administration. Although recognized, there was no increase in IgA and IgG by PSF-2 and CTB, and no immune enhancing effect was observed. On the other hand, in the case of subcutaneous injection, a significant increase in IgG and a clear increase in IgA were observed due to the split influenza vaccine administration, but again there was no increase in antibody production by PSF-2 and CTB, and the immune enhancement effect was I was not able to admit. Particularly in the case of subcutaneous injection, IgG was specifically increased in blood. Similar results are obtained when PSF-1 is used instead of PSF-2 (not shown).
Even if PSF-2 and CTB were administered nasally and subcutaneously at the time of primary immunization and secondary immunization, respectively, no increase in virus-specific IgA and IgG in the blood was observed, and PSF-2 and CTB The effect was determined to be an effect of enhancing vaccine action (not shown in the figure).
Example 5
Nasal administration (a), subcutaneous injection (b) Effects of PSF-2 and CTB on antigen presentation ability of nasal, lung and splenic dendritic cells by influenza vaccine
As a nasal vaccine, 0.1 μg of a split influenza vaccine was administered alone, or together with 0.1 μg of PSF-2 and 0.1 μg of CTB, a total of 2 μl was administered to both noses of BALB / c mice as a 1 μl PBS solution. As a subcutaneous vaccine, the same amount of split influenza vaccine, PSF-2, and CTB as the nasal vaccine were administered subcutaneously to the neck of the BALB / c mouse as a 50 μl PBS solution. Two days later, the mice were sacrificed, dendritic cells were prepared from the nose, lungs, and spleen, and the cell surface expression levels of MHC class II, CD40, B7-1 (CD80), and B7-2 (CD80) were measured by flow cytometry. did.
As a result, increased expression of antigen presentation-related molecules CD40 and B7-2 on the membrane surface of nasal dendritic cells (antigen-presenting cells) challenged with vaccine by CTB was confirmed, and the adjuvant effect was confirmed at the molecular level. . In the case of PSF-2, in addition to CD40 and B7-2 of nasal dendritic cells, increased expression of MHC II molecules is also observed, and at least three molecules of dendritic cells are involved in the immune enhancing effect. It became clear.
However, no clear changes could be observed in the CD40, B7-2 and MHC II molecules of lung and spleen dendritic cells. Moreover, even if PSF-1 and PSF-3 were used instead of PSF-2, a slight effect was obtained. The same phenomenon was confirmed for PSF-4 and -5, but the effect was attenuated.
Example 6
Effects of SPF-2 and CTB on TGF-β1 secretion level in nasal cavity (a) and alveolar (b) mucosa by nasal influenza vaccine administration
As a nasal vaccine, 0.1 μg of a split influenza vaccine was administered alone, or together with 0.1 μg of SPF-2 and 0.1 μg of CTB, a total of 2 μl was administered as a 1 μl PBS solution to both noses of BALB / c mice. As a subcutaneous vaccine, the same amount of vaccine as nasal vaccine, PSF-2, and CTB were administered subcutaneously to the neck of the BALB / c mouse as a 50 μl PBS solution. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. Two weeks after the second immunization, the mice were sacrificed, and nasal washes were prepared and used to quantify TGF-β1 secretion (n = 15-20; mean ± SE; ^* The significance level by t test is p <0.01 for the vaccine administration group.
In order for B cells to differentiate into IgA-producing cells (class switch), it is known that the local TGF-β1 concentration in which the producing cells are localized is important (Stavzer, J .: Regulation of antibody). production and class switching by TGF-beta.J.Immunol.155 (4), 1647-1651, 1995). Therefore, the concentration of TGF-β1 in the nasal cavity (a) and alveoli (b) mucosal region under the above conditions was examined.
Both the nasal cavity and alveolar mucosa local TGF-β1 concentrations administered with the split influenza vaccine increased significantly in the presence of SPF-2 and CTB. The degree of increase was not significantly different between SPF-2 and CTB. The results are shown in FIGS. 5 (a) and 5 (b). In vivo SPF-2 was found to increase the concentration of TGF-β1 that promotes the promotion of IgA-secreting B cell differentiation to the same extent as the foreign toxin CTB. Although not shown in the figure, even if PSF-1 or PSF-3 is used instead of PSF-2, a slight effect is obtained. The same phenomenon was confirmed for PSF-4 and -5, but the effect was reduced. Even if PSF-2 and CTB are administered nasally or subcutaneously during the first and second immunizations, respectively, no increase in TGF-β1 concentration is observed, and the effect of PSF-2 and CTB is the vaccine. It was determined as an effect of enhancing the action (not shown in the figure).
Example 7
Effects of PSF-2 and CTB on production of anti-influenza specific antibodies (IgA, IgG) in nasal cavity (a), alveoli (b) and blood (c) by intranasal influenza vaccine
As a nasal vaccine, 0.2 μg of split influenza vaccine alone, or PSF-2 0.2 μg as an AD vehicle, or CTB 0.2 μg as an adjuvant, and 1 μl of a total of 2 μl administered as a PBS solution to both noses of BALB / c mice It was. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. In each group, mice were sacrificed 2 weeks after the secondary immunization, and nasal lavage fluid, alveolar lavage fluid, and serum from heart blood were prepared, and these were used to quantify the expression level of anti-influenza antibodies (n = 15-30). , Mean ± SE, +, p <0.01 vs. vaccine alone).
As shown in FIGS. 6 (a) and 6 (b), in the nasal lavage fluid and alveolar lavage fluid, blood anti-influenza antibody IgA (blue circle) showed a marked increase when PSF-2 and vaccine were administered. However, as shown in FIG. 6 (c), no increase in IgG (red circles) was observed in the blood.
On the other hand, when CTB and vaccine were administered, IgG and IgG increased in the nasal and alveolar lavage fluids (FIGS. 6 (a) and (b)) and also significantly increased in blood (FIG. 6 (c)). ).
As described above, in the case of CTB, a systemic immune response (systemic immune response) was generated in addition to establishing local immunity in response to a nasal inoculated antigen, as previously reported. On the other hand, when PSF-2 was used, only local mucosal immunity was established.
Note that J. et al. Freek van Iwaarden et al. (Non-patent Document 4) can induce a systemic immune response with SP-B and lipids when artificially removing macrophages from the lung, but induce immunity when macrophages are not removed. Has reported that it is not possible. Moreover, in the said literature, SP-B + lipid amount required in order to induce | guide | derive a predecessor immune reaction is 250-300microliter, and is far from the PSF-2 dosage (0.2microliter) of the said Example. . Moreover, no mention is made of local mucosal immunity.
Example 8
Effects of SPF-2 and CTB on various cytokines secreted from nasal, lung and splenic lymphocytes by nasal influenza vaccine administration
As a nasal vaccine, 0.2 μg of split influenza vaccine was administered to the upper respiratory tract of BALB / c mice in a total amount of 2 μl together with SPF-20.2 μg or CTB 0.2 μg as a 1 μl PBS solution. Two weeks after the second immunization, the mice were sacrificed, and the secreted amounts of TGF-β1 and cytokines (IL-4, IL-5, IL-6, IL-13) from nasal cavity, alveoli and spleen lymphocytes were determined. (N = 15-20; mean ± SE; +++, P = 0.06; ++, P = 0.05; +, P = 0.01 vs. vaccine alone).
As shown in FIGS. 7 (a) to (e), significant increase in secretion of TGF-β1, IL-5 and IL-6 was observed in the mucosal region (nose, lung) after immunization with PSF-2. However, no significant increase was observed in IL-4 and IL-13. On the other hand, no significant increase in any cytokine was observed in the spleen.
From the above results, it was confirmed that PSF-2 increases Th2 type cytokines that promote differentiation and induction of B cells that produce IgA locally in the mucosa.
Example 9
Effect of PSF-3 on production of anti-influenza specific antibodies (IgA, IgG) in nasal cavity (a), alveoli (b) and blood (c) by intranasal influenza vaccine
As a nasal vaccine, 0.2 μg of a split influenza vaccine alone, or as an AD vehicle, PSF-3 (SP-B fragment 253 to 278 peptide described in SEQ ID NO: 16 and SP-C fragment 24 to 58 described in SEQ ID NO: 21) Equal-weight mixture with peptide) 0.2 μg or lipid component 0.2 μg was administered as a PBS solution to BALB / c mice on both noses, 1 μl in total, 2 μl. After 4 weeks, secondary immunization was performed by the same method as the primary immunization. Each control group received the same volume of PBS. In each group, mice were sacrificed 2 weeks after the secondary immunization, and nasal lavage fluid, alveolar lavage fluid, and serum from heart blood were prepared, and these were used to quantify the expression level of anti-influenza antibodies (n = 15-30). , Mean ± SE, +, p <0.01 vs. vaccine alone).
As shown in FIGS. 8 (a) and (b), in the nasal lavage fluid and alveolar lavage fluid, blood anti-influenza antibody IgA (blue circle) showed a marked increase when PSF-3 and vaccine were administered. However, no significant increase in IgG (red circles) was observed. On the other hand, in serum (c), neither IgG (red circle) nor IgG (blue circle) significantly increased.

The “AD vehicle” according to the present invention exerts the function of transporting all substances such as antigens, drugs, nutrients, etc. from the mucous membrane of the nose, trachea, intestinal tract, etc. or the skin into the cells, and gives priority to IgA production there. Therefore, it enables mucosal vaccine, allergy prevention and treatment, transmucosal and transdermal DDS, transmucosal administration and transdermal administration of useful substances such as drugs and nutrients. Moreover, its clinical use and safety have already been approved in Japan and abroad.
Therefore, biopharmaceuticals, pharmaceuticals and pharmaceuticals in DDS, food and drinks and food industry in functional foods and health foods, agriculture, agricultural chemicals, fish diseases in the cultivation, cultivation, disease control, insect control, etc. of agricultural products It is expected to be used and utilized in a very wide range of industries, such as cultivation and fisheries for vaccines and medications, construction work for ants and insects, and environmental protection.

Claims (6)

At least one fragment selected from lung surfactant protein B or a plurality of fragments derived from protein B, at least one fragment selected from lung surfactant protein C or a plurality of fragments derived from protein C, and at least one lipid The protein B fragment is a peptide consisting of the amino acid sequence of SEQ ID NOs: 2, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The protein C fragment is obtained by coexisting, contacting, capturing, or adsorbing an antigen to an antigen drug vehicle that is a peptide consisting of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 9, 21, or SEQ ID NO: 6. An intranasal mucosal vaccine characterized by inducing mucosal immunity by promoting production of IgA antibodies in the mucosal region. At least one lipid is phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylserine, dipalmitoylglycerophosphocholine, diacylglycerophosphoglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, lauric acid, myristic acid, palmitic acid, stearin The mucosal vaccine according to claim 1, which is selected from a lipid group consisting of an acid and oleic acid. The lipid is a sheet-like or roll-like membrane, and is composed of at least one fragment selected from lung surfactant protein B or a plurality of fragments derived from protein B, and lung surfactant protein C or a plurality of fragments derived from protein C The mucosal vaccine according to any one of claims 1 and 2, wherein each of a plurality of chains of at least one selected fragment is spiked in a state where a hydrophobic region end is inserted into the lipid membrane. The mucosal vaccine according to any one of claims 1 to 3, wherein the induction of mucosal immunity is characterized by promotion of production of TGF-β1 and Th2-type cytokines in the mucosal region. A method for inducing mucosal immunity, comprising administering the mucosal vaccine according to any one of claims 1 to 4 to the nasal cavity of a non-human animal to promote production of IgA antibody in the mucosal region. 6. The method for inducing mucosal immunity according to claim 5 , wherein the induction of mucosal immunity is characterized by promotion of production of TGF-β1 and Th2-type cytokines in the mucosal region.

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